Thrombospondin (also known as thrombin sensitive protein or TSP) is a 450,000 molecular weight protein composed of three identical disulfide-linked polypeptide chains (Lawler et al. J. Cell Biol (1986) 101:1059-71). TSP is secreted by platelets in response to physiological activators such as thrombin and collagen (Lawler, J. Blood (1986) 67:112-123). TSP comprises 3% of the total platelet protein and 25% of the total platelet alpha granular protein (Tuszynski, G. P. et al. (1985) J. Biol. Chem. 260:12240-12245). Other cells also synthesize TSP including fibroblasts (Jaffe, E. A. et al., (1983) Natl. Acad. Sci. USA 80:999-1002), smooth muscle cells (Raugi, G. J. et al., (1982) J. Cell Biol. 95:351-354), and endothelial cells (McPhearson, J. et al. J. Biol. Chem. 256:11330-11336). TSP has been found in certain tumor tissues, such as melanoma cells (Varani, J. et al., (1989) Clin. Expl. Metastais 7:319-329), squamous lung carcinoma (Riser, B. L. et al., (1988) Exp. Cell Res. 174:319-329) and breast carcinoma (Pratt, D. A. et al., (1989) Eur. J. Cancer Clin. Oncol. 25:343-350). In addition, the following tumor cells in culture have been shown to synthesize TSP: fibrosarcoma, rhabdomyosarcoma, glioblastoma, Wilm's tumor, neuroblastoma, teratocarcinoma, choriocarcinoma, melanoma, and lung carcinoma (Mosher, D. F., (1990) Annu. Rev. Med. 41:85-97). A number of recent studies have shown that TSP plays a major role in cell-cell and cell substatum adhesion (Tuszynski, G. P. et al., (1987) Seminars in Thrombosis Hemostasis (13:361-368, Mosher, D. F., (1990) Annu. Rev. Med. 41:85-97). TSP promotes cell attachment, platelet aggregation, and lung tumor colony formation in a murine model of experimental metastasis (Tuszynski, G. P. et al., (1987) Science 236:1570-1573, Tuszynski, G. P. et al., (1988) Blood 72:109-115). The role of TSP in adhesion is further supported by the observation that the extracellular matrix of most tissues contains TSP.
TSP is composed of linear polypeptide domains that specifically interact with various macromolecules such as plasma and matrix components. For example, TSP forms a complex with heparin (Yabkowitz, R. et al. (1989) J. Biol. Chem. 264:10888-10896), fibrinogen (Tuszynski, G. P. et al. (1985) J. Biol. Chem. 260:12240-12245), collagen (Mumby, S. M. et al. (1984) J. Cell Biol. 98:10888-10896, and plasminogen (Depoli. P. et al. (1989) Blood 73:976-902). The structure of TSP is conserved among various animal species as indicated by the fact that the antiobody against the human protein cross-reacts with TSP from mouse, rat, pig, cow, sheep, dog, and turkey (Switalska, H. I. et al., J. Lab Clin. Med. 106:690-700).
Thrombospondin has been purified by a number of procedures including exclusion chromatography (Lawler et al., J. Biol. Chem. (1978) 253:8609-16), heparin affinity chromatography (Lawler et al., Thromb. Res. (1981) 22:267-269), fibrinogen affinity chromatography (Tuszynski et al., J. Biol. Chem. (1985) 260:12240-5), barium chloride precipitation (Alexander et al., Biochem. J. (1984) 217:67-71) and anion exchange chromatography with HPLC (Clezarolin et al., J. Chromatog. (1984) 296:249-56).
The complete amino acid sequence of TSP has been deduced from DNA clones prepared by various groups including Lawler et al., J. Cell Biol. (1986) 103:1635-48; Kobayashi et al., Biochemistry (1986) 25:8418-25; Dixit et al., Proc. Ntl. Acad. Sci. (1986) 83:5449-53; and Hennessy et al., J. Cell Biol. (1989) 108:729-36.
Cell adhesion is critical to the development and survival of multicellular organisms. The process of cell adhesion is complex requiring numerous extracellular proteins such as fibronectin, vitronectin, collagen, laminin, and TSP and numerous families of cellular receptors such as the integrins and cellular adhesion molecules (CAMS). These molecules are involved in the adhesion of both normal and tumor cells and have been studied quite intensively in recent years.
The amino acid sequence, Arg-Gly-Asp (RGD), was established as a cell attachment domain in fibronectin (Pierschbacher, M. D. and Ruoslahti, E., (1984) Nature (London) 309:30-32). The same or related sequences have been discovered in many proteins and serve as cell binding sites for such macromolecules as fibrinogen (Ginsberg, M. D. et al., (1985) J. Biol. Chem. 260:11891-11896). However, it appears that the adhesive function of laminin may not be based on the RGD sequence, but on a peptide segment of the Bi chain containing the amino acid sequence tyrosine-isoleucine-glycine-serine-arginine (YIGSR) (SEQ ID NO: 33) (Sasaki, M. 1987, Proc. Natl. Acad. Sci. 84:935-938). Synthetic peptides containing the RGD and YIGSR (SEQ ID NO: 33) sequence promote cell adhesion.
The therapeutic use of synthetic peptides based on the adhesive domains of fibronectin and laminin have recently been reported. Humphries et al. (2986) Science 233:467-470) were the first to demonstrate that co-injection of the pentapeptide GRGDS with B16-F1O murine melanoma cells dramatically inhibited the formation of lung colonies in C57BL/6 mice. Another synthetic peptide which was derived from laminin (YIGSR) (SEQ ID NO: 33) also dramatically inhibited B16-F1O melanoma cell metastasis in C57B1/6 mice (Kleinman, H. K. et al., (1987) Science 238:1132-1133; Kleinman, H. K. et al., (1990) Proc. Natl. Acad. Sci. USA 87:2279-2283). The inhibitory activity of these peptides may be due to competition with endogenous laminin and fibronectin-dependent adhesion of tumor cells to the vascular bed of the target organ during the metastatic dissemination of the tumor cells.
Because metastasis is a step-by-step process involving the transfer of tumor cells from one site to another through the lymphatic and blood circulation and platelet. reduction in animals effectively blocked metastasis in animals (Gasic et al, (1968) Proc. Natl. Acad. Sci. USA 48:46-52), platelets have been thought to play a special role in the development of metastasis. Since TSP comprises 25% of the total alpha granular platelet secreted-protein, TSP would be expected to have a major role in the hemotagenous transfer of tumor cells to distant organs. Indeed, TSP has been shown to promote tumor cell metastasis in a murine model (Tuszynski et al, (1987) 47:4130-4133). In addition, events which accompany platelet activation, such as: secretion of adhesive proteins, platelet aggregation, activation of proteolytic enzymes, and activation of the clotting cascade have all been shown to play a significant role in tumor cell metastasis (Gasic, G. J., (1984) Cancer Metastasis Rev. 3:99-116).
Adhesive proteins which are part of the extracellular matrix control the movement, growth, and morphology of many cell types. Extracellular matrix proteins interact with tumor cell receptors and affect tumor cell adhesion to basement membrane collagen in different ways. For example, exposure of melanoma cells in vitro to laminin resulted in increased capacity of tumor cells to adhere to the basement membrane and to produce lung tumor colonies (Barsky, S. H. et al., (1984) J. Clin. Inv. 74:843-848; Terranova, V. P. et al., (1984) Science 226:982-985).
In view of the information described above, TSP may play an important role in many diverse and clinically important processes, such as: cell migration, wound healing, nerve regeneration, and tumor cell metastasis. To better understand the pathophysiology of these processes at the molecular level, assignment of each of the biological activities of TSP to a specific subdomain or oligopeptide of TSP would provide valuable information. Specifically, detailed knowledge of the structure of domains of the TSP and TSP receptors could be used to design TSP antagonist peptides which could block pathophysiological activities of TSP such as TSP-dependent tumor cell metastasis formation.
TSP contains three homologous peptide sequences designated type I, II, and III repeats (Lawler and Hynes, (1986) J. Cell Biol. 103:1635-1648). The three repeats consist of approximately 60 amino acids each containing six cysteine residues. Type I repeats exhibit homology with peptide segments found in a number of diverse proteins. We have identified two hexapeptide sequences in TSP (CSTSCG (SEQ ID NO: 7) and CSVTCG (SEQ ID NO: 2)) that are either totally conserved in other proteins or present with one or two conservative amino acid substitutions. The prevalence of the conserved sequences is indicated in Table I below.
TABLE I Protein Sequence Reference TSP CSVTCG Lawler et al., (1986), (SEQ ID NO: 20) J. Cell Bio/. 103: 1635-48 CSTSCG (SEQ ID NO: 14) Circumsporozoite CSVTCG Dame J. B. et al., (1984) (SEQ ID NO: 2) Science 225: 593-599 Trap CSVTCG Robson K. J. H. et al., (1988) (SEQ ID NO: 2) Nature 335: 79-82 Properdin CSVTCG Goundis, D. et al. (1988) (SEQ ID NO: 2) Nature 335: 82-85 Glycoprotein E CVVTCG McGoech D. J. et al, (1985) Herpes simplex I (SEQ ID NO: 15) J. Mol. Biol. 181: 1-13 Cytochrome C CSETCG Lawson, J. E. et al., (1985) oxidase (SEQ ID NO: 16) Curr. Genet. 9: 351-360 polypeptide II Respiratory CSVTCK Blasco, F. et al. (1989) nitrate reductase (SEQ ID NO: 9) Mol. Gen. Genet. 218: 249-256 beta chain Bird spider 18S CSVSCG Hendriks L. et al., (1989) ribosomal RNA (SEQ ID NO: 17) Eur. J. Biochem. 177: 15-20 Chicken alpha CSVVCG Lemischka I. R. et al. (1981) tubulin (SEQ ID NO: 18) J. Mol. Biol. 151: 101-120 Zebrafish CSKTCG Njolstad P. R. et al., (1990) homeobox gene (SEQ ID NO: 19) EMBO J. 9: 515-524 E. coli gut CSVTCX Yamada M. et al., (1987) operon (SEQ ID NO: 20) Biol. Chem. 262: 5455-5463 E. Coli ATPase CSVTCM Kanazawa H. et al., (1981) (SEQ ID NO: 21) BBRC: 103: 613-620 Rat liver CSVGCG Poncin J. E. et al., (1984) apolipoprotein A-I (SEQ ID NO: 22) Eur. J. Biochem. 140: 493 Tryptophan CWVTCG Brosius, J. et al., (1982) synthetase (SEQ ID NO: 23) Gene 17: 223-228 Highlands J virus CSVTCL Ou J. H. et al., (1982) (SEQ ID NO: 24) J. Mol. Bioi. 156: 719-730 Human c-myb CSVTCK Slamon D. J. et al., (1986) proto-oncogene (SEQ ID NO: 9) Science 233: 347-351 Antistasin CRKTCP Ginsberg V. et al. (1990) (SEQ ID NO: 25) J. Biol. Chem. 264: CRVHCP 12138-12140 (SEQ ID NO: 26) EtplOO CVCECG Tomley F. et al (1989) (SEQ ID NO: 27) 5th International Coccisiosis CSATCG Conference 469-573 (SEQ ID NO: 28) CSRTCG (SEQ ID NO: 11) CSEQCG (SEQ ID NO: 29) Human desmin CSVTCH Li Z. et al., (1989) (SEQ ID NO: 30) Gene 78: 243-254 Human related CSVPCG May F. E. B. et al., J. Virol mammary tumor (SEQ ID NO: 31) 60: 743-749 virus Human proto- CSRTCG Ouweland A. M. W. et al., oncogene c-fes/fps (SEQ ID NO: 11) EMBO J. 4: 2897-2903 Platelet GPIIb CSVTCR Bray P. F. et al., (1987) (SEQ ID NO: 10) J. Clin. Invest. 80: 1812-1817
The present invention provides thrombospondin fragments and analogs which mimic or inhibit the biological activity of intact thrombospondin which find use in a variety of biological, prophylactic or therapeutic areas.